WO2004087923A1

WO2004087923A1 - A NEW STRESS-RESISTANCE TRANSCRIPTION FACTOR (CaPIF1 AND NbPIF1), ITS GENE AND A STRESS-RESISTANCE PLANT TRANSFECTED BY THE GENE

Info

Publication number: WO2004087923A1
Application number: PCT/KR2003/001936
Authority: WO
Inventors: Doil Choi; Sang-Keun Oh; Young Hee Joung; Sanghyeob Lee; Jeong-Mee Park
Original assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Current assignee: Korea Research Institute of Bioscience and Biotechnology KRIBB
Priority date: 2003-03-31
Filing date: 2003-09-23
Publication date: 2004-10-14
Anticipated expiration: 2005-09-30
Also published as: AU2003264961A1; KR100586084B1; KR20030031096A

Abstract

The present invention relates to CaPIF1, pathogen-inducible pepper transcription factor, having plant specific TFIII A-type C2H2 zinc finger domain motif isolated from pepper cDNA library. More particularly, the present invention relates to a DNA encoding plant stress resistant inducible transcription factor having C2H2 zinc finger motif or a DNA capable of hybridizing said DNA, a plant stress resistant inducible transcription factor protein encoded said DNA, an expression vector for plant transformation capable of providing plants with stress resistance because of said DNA contained, plant cells transformed with said expression vector, stress resistant plants or seeds transformed with said expression vector and the method for preparing of stress resistant plants, in which said DNAs are introduced into plants. According to the present invention, useful plants, which have resistance to various biotic or abiotic stresses, can be bred by simple and systematic method.

Description

A NEW STRESS-RESISTANCE TRANSCRIPTION FACTOR (CaPIFl and

NbPIFl), ITS GENE AND A STRESS-RESISTANCE PLANT

TRANSFECTED BY THE GENE

FIELD OF THE INVENTION

The present invention relates to a pathogen-induced hot pepper (Capsicum annuum) transcription factor, CaPIFl (Capsicum annuum athogen-Jnducible /factor 1) containing the plant specific TFIIIA-type C2H2 zinc finger domain motif isolated from the hot pepper cDNA library and a tobacco transcription factor, NbPIFl (Nicotiana benthamiana Pathogen — Induced Factor 1). The present invention also relates to genes encoding the above transcription factor and the stress resistant plant transformed with the above genes.

BACKGROUND OF THE INVENTION

Plants defend themselves from pathogens by activating various defense mechanisms. One of the most rapid responses raised by pathogen attack is the hypersensitive response(HR), which is characterized by rapid and localized cell death at the site of pathogen invasion (Lamb et al., 1989; Dangl et al., 1996). The rapid cell response is often caused by avirulent pathogen recognition and activity of highly controlled signal transduction pathway (Martin, 1999; Ellis, 2000). Such responses induce a plant immune response known as SAR (systemic acquired resistance)(Yang et al., 1997). SAR is characterized by enhanced resistance to toxic pathogen and is accompanied by the defense response including transcription activity of defense related genes, accumulation of antibacterial compound and pathogenesis-related (PR) proteins and strengthening cell wall (Kuc, 1982; Ward et al., 1991; Uknes et al., 1992; Dixon et al., 1996).

The plant signaling molecules, such as salicylic acid (SA), ethylene (ET), and jasmonic acid (JA), that accumulate in plants during pathogen infection have been known as principle components for defense response pathway (Dong, 1998; Dempsey et al., 1999; Reymond and Farmer, 1998; Pieterse and van Loon, 1999; Penninckx et al., 1998). Many PR genes are regulated by one or more of the signaling molecules which are increased in plants as a result of pathogen infection (Malamy et al., 1990; Thomma et al., 1998). The SA dependent or JA/ET-dependent defense-signaling pathways regulate the expressions of acid-PR proteins (Rl, PR2 and PR-5) or alkaline-PR proteins (PR-3, PR-4 and PDF1.2.). Many studies have reported that various genetic and biochemistrical phenomena are present for communications between different pathways, that are capable of cooperative regulation of antagonistic responses (Maleck and Dietrich, 1999; Feys and Parker, 2000). Furthermore, SA dependent and/or JA/ET-dependent defense-signaling pathways show pathogen specific and/or pathogen dependent (Thomma et al., 1998). It is needed cooperative operations of transcription factors by the regulation pathways and there are shown various transcriptional levels of many transcription factors at each stage of development or under various stress environments such as biotic or abiotic treatment (Solano et al., 1998; Sakamoto et al., 2000; Liu et al., 1998; Eulgem et al., 1999; Park et al., 2001; Chen et al., 2002). Plants show characterized stress responses that are against various biotic stresses. Recently, it was reported that the transcription factors of some classes related to defense responses of plants are DNA-binding proteins having AP2/EREBP domain (PH4/5/6, Zhou et al., 1997; Gu et al., 2002; He et al., 2001; Tsil, Park et al., 2001), bZIP domain (TGA bzip), Myb domain (R2R3 AtMYB30, Vailleau et al., 2002) and WRKY zinc-fmger domain (AtWRKYό, Robatzek and Somssich, 2002; AtWRKY 18, Chen and Chen, 2002), that are shown in some plants.

Some classes of the zinc- finger motifs are present in transcription factors and function as components of DNA-binding domain (TFIIIA and GATA type) and protein-protein interaction domain(Lim-, PHD-finger and RLNG-finger type)(Takatsuji, 1998; 1999). TFIIIA(C2H2)— type zinc fingers were identified in many eukaryotic transcription factors. In plants, it has been known that most of these proteins participate in the regulation of specific and important biotic processes such as reproduction organ formation, leaves initiation, lateral shoot generation, gametogenesis, seed or pollen development (Dinkins et al., 2002; Kapoor et al., 2002; Takatsuji. 1999) and the resistance to abiotic stress (Frugier et al., 2000; Kim et al., 2001; Lippuner et al., 1996). The first TFIIIA-type zinc-finger protein in plants was identified as DNA-binding protein PEThy;ZPT2-l (this protein was denoted EPF1) in Petunia which is regarded to regulate gene expression of 5- enolpyruvyshikimate-3-phosphate synthase^VSPS), petal specific enzyme which participates in shikimate pathway for anthocyanin production, floral pigment(Takatsuji et al., 1992).

EPF family is a group of DNA-binding protein forming C2H2 zinc finger motif(CX2CX3FX5LX2HX3H) that is believed to function in the reproductive organ-specific transcriptional regulation of Petunia. SCOF-1 gene is low temperature and ABA-induced soybean zinc finger protein. Constitutive overexpression of SCOF-1 in Arabidopsis induces expression of COR gene and increases the resistance to low temperature of transgenic tobacco and Arabidopsis (two finger type, Kim et al., 2001). AlZFPIO (A. thaliana single zinc finger protein) overexpressing tobacco showed dwarfishness, phenotypic abnormalities of leaves and early flowering in relation to the level of expression of AtZFP 10 (Dinkins et al., 2002). The ectopic(abnormal position) expression of superman^c/P) gene in tobacco suppressed generation of petals and stamens(Yun et al., 2002: one finger type). TAZl (Tapetum developmwnt zinc finger protein 1: three finger type) was isolated from the anther of Petunia. Cosuppression of TAZl induced abnormal generation and early degeneration of tapetum (Kapoor et al., 2002). Broad microarray analysis of gene expression provided additional evidence that C2H2 zinc finger proteins were induced or decreased after pathogen infection in Arabidopsis (Chen et al., 2002. supplemented data). However, it is not clear what the role of transcription factor, TFIIIA-C2H2-type zinc finger, is during pathogen infection.

Many C2H2 zinc finger genes encoding C2H2 zinc finger domain and QALGGH sequence which have been in several plants are induced during development of genital tissues (Takatsuji, 1998) or by abiotic stresses such as wounds, cold and salt treatment (van der Krol et al., 1999). ZPT2-1 of petunia interacts with the promoter region of the 5-enolpyruvyshikimate-3- ρhosphate(EPSPS) gene (Takatsuji et al., 1992). It has been found that WZF1 of wheat is a DNA-binding protein, particularly interacting with a cis-element of histone genes (Sakamoto et al., 1993). Regulatory functions have been assigned to TFIIA-type zinc finger proteins in Arabidopsis, petunia and Chinese cabbage and soybean. SUPERMEN, AtZFPl, PetSPL3 and BcZRPl have been implicated in the developmental regulation of various floral and vegetative organs, presumably through the control of cell division and/or expansion in particular cell types. (Takatsuji, 1999). STZ (Lippuner et al., 1996) and SCOF-1 (Kim et al., 2001) participate in response or resistance of plants to various environments such as salt or cold stress.

DISCLOSURE OF THE INVENTION

Accordingly, an object of the present invention is to provide a novel transcription factor and the gene for the factor, which give a plant stress resistance.

Another object of the present invention is to provide a plant transformed with the gene for the novel transcription factor, which gives a plant stress resistance.

To achieve the above objects, the present invention is based on the fact that a novel gene, CaPIFl (Capsicum annuum Pathogen-Jnducible /factor 1, two zinc- finger type) encoding putative TFIIIA-type C2H2 zinc finger protein was isolated from hot pepper cDNA library(refer to http://plant.pdrc.re.kr ) and characterized. Moreover, the present invention provides a DNA encoding stress-resistance inducible transcription factor having more than 80% of homology with amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

In the present invention, the DNA is preferably CaPIFl gene having the base sequence of SEQ ID NO: 1, NbPIFl (Nicotiana benthamiana Pathogen Inducible Factor 1-like, SEQ ID NO: 2) gene having the base sequence of SEQ ID NO: 2 or a DNA capable of hybridizing with said genes.

In the present invention, the stress includes both biotic stress such as viral, bacterial, fungal pathogen and abiotic stress such as wound, cold, chemicals.

Furthermore, the present invention provides a plant stress resistant inducible transcription factor protein, which is encoded by said each DNA.

Said transcription factor proteins have C2H2-type zinc finger motif, in common and are preferably CaPIFl protein having the base sequence of SEQ ID NO: 3 or NbPIFl protien having the base sequence of SEQ ID NO: 4.

In addition, the present invention provides expression vectors containing said each DNA, plant cells transformed with said expression vector, stress resistant plants or its seeds transformed with said expression vector.

The present invention also provides a method for preparing of stress resistant plants, in which said various DNAs or expression vectors are introduced into plants and the method of regulating stress resistance of plants in which expression of plant stress resistant inducible transcription factor protein is changed.

Hereinafter, the present invention will be described in detail.

According to the present invention, it has been found that transcripts of CaPIFl genes are derived from leaves treated with salicylic acid (SA), ethephon or jasmonic acid (JA) as well as tissues infected by host-incompatible and nonhost- incompatible pathogens. According to the expression analysis using CaPIFl ::smGFP fusion gene, CaPIFl protein is a cell nuclear protein transferred to the cell nucleus and the fusion protein of LexA DNA binding domain and CaPIFl activates transcription in yeast. Over-expression of CaPIFl shows resistance to bacterial pathogens and always induces expression of PR — or defense-related genes. Therefore, plants transformed with CaPIFl showed diseases resistance reaction to bacterial pathogens. Also, in experiments using CaPIFl orthologues as the subject, the silencing of the orthologues increased disease infection by bacterial pathogens. From these results, it is presumed that C2H2 — type zinc-finger CaPIFl transcription factor is preferentially induced by attack of pathogens and CaPIFl gene is involved in the signal transduction pathways of the defense response of plants.

Now, various properties of CaPIFl will be explained in detail hereinafter.

(1) CaPIFl is a nuclear factor containing TFIIIA-type C2H2 zinc finger

In the present invention, 90 candidates for a transcription factors were sorted out from the pepper EST sequence DB (http://plant.pdrc.re.la') and analyzed for expression pattern by the microarray method through reverse northern blot analysis with Xanthomonas axonopodis pv. glycines 8ra which is a non-host bacterial pathogens in pepper.

According to the present invention, it has been identified that CaPIFl is a new transcription factor containing the C2H2-type zinc finger domain in pepper

Capsicum annuum L. CaPIFl comprises two canonical C2H2-type zinc finger motifs (CX2CX3FX5LX2HX3H) and the conserved QALGGH sequence

(Takatsuji, 1998; Kubo et al., 1998).

Judging from the whole structure, CaPIFl is most similar to petunia EPF family having two C2H2-type zinc finger motifs between which a sequence of about 38 amino acids are particularly PetZFP4 (corresponding to 77% amino acid) (Takatsuji et al., 1994). The CaFIFl protein also has an alkali region considering the function of nuclear localization signal around N- (³⁵KGKRSKRSR⁴³) and C- (¹⁹⁴PAKKSRL²²⁰) ends.

According to the present invention, it has been identified that the CaPIFl protein is a nuclear protein transferred to the nucleus.

In the present invention, some transformed tobacco plants expressing

CaPIFl in the sense orientation were obtained and TI transformed plants were selected therefrom, based on northern blot analysis and kanamycin resistance.

However, the transformed plants did not show a remarkable morphologic phenotype at all.

Since STZ and SCOF-1 increased resistance to salt and cold of the transformed plants, an experiment was performed to see if 35S::CaPIFl is affected by these stresses. The expression of the CaPIFl gene was increased by salt treatment (400mM, feeding method) and 35S::PIF1 is not significantly shown under salt and cold treatments.

(2) Over-expression of CaPIFl in tobacco provides resistance to bacterial pathogens

In the present invention, in vivo functions of CaPIFl in the defense mechanism were identified through the expression of the CaPIFl gene in tobacco plants. The over-expression of CaPIFl also strengthens resistance to bacterial pathogens in the tobacco plants. It is presumed that such phenomena are caused by the increased expression of PR- or defense-related genes induced by the over- expression of CaPIFl, as described below.

Also, the increase of the resistance to bacteria has been shown in plants, in which other transcription factors including AP2/EREBP family (Pti5, He et al., 2001; Pti4, Gu et al., 2002; Tsil, Park et al., 2001), Myb (R2R3AtMYB30, Vailleau et al., 2002) and WRKY zinc-fmger (AtWRKYό, Robatzek and Somssich, 2002; AtWRKY 18, Chen and Chen, 2002) and the like are over-expressed. The expression of the above-described genes in plants increased the expression level of the SA- or ET/JA-regulated gene. The SA- or JA/ET-dependent defense signal pathways regulate the expression of acid (PRl, PR2 and PR-5)- or alkalic (PR-3, PR4 and PDF1.2.)-PR protein. Though the tobacco family expressing CaPIFl showed strengthened resistance to bacterial pathogens, the CaPIFl gene is induced by the SA or ET/JA treatment and always expressed any one of PRl, PR2 (SA-dependent) gene or PR3, PINII (ET/JA-dependent) gene. From these observations, it can be concluded that the CaPIFl gene plays an important role in the communication between these pathways.

(3) Over-expression of CaPIFl induces the activation of defense-related gene.

According to the present invention, the over-expression of CaPIFl induces constitutive defense response.

According to RNA blot analysis, it was shown that 35::CaPIFl constantly expresses molecules which serve as a marker in the defense response. Even under non-stress conditions, the over-expression of the CaPIFl gene in transformed tobaccos activates the expression of PR genes such as PRl, PR2, PR3, PR5, SAR8.2, Peroxidase, PINII, PAL and the like. It has been well known that a certain transcription factor serves as a target for another signal pathway.

This fact has been proved by the microarray analysis, in which mRNA corresponding to 28 of 1475 marked cDNAs expressed in a young seedling was expressed much higher enough to show statistic significance in 35S::CaPIFl than inpMBPl. It has been lαiown that the expression of numerous defense-related genes including PR gene is activated by various biotic or abiotic stresses such as (D viral, bacterial or fungal pathogens (2) treatment of plant hormones and elicitors such as ethylene, SA, JA, H₂O₂ and UV rays (Bol et al., 1990; Linthorst, 1991) (3) ectopic expression of foreign genes (Chen and Chen 2002; Gu et al., 2002; Park et al., 2001; Zhou et al., 1997). Though the expression of PR genes is usually induced by the infection of pathogens, the expression is not always enhanced by the ectopic expression of the foreign genes.

In the present invention, it has been proved that the expression of the PR genes in tobacco is strengthened by the over-expression of the CaPIFl gene. Therefore, the gene set involved in the pathogen protection and SAR, which is considered to be regulated by the shared CaPIFl transcription factor, is always expressed in CaPIFl transformed plants. However, it is not clarified if CaPIFl works directly and/or indirectly.

(4) Silencing of CaPIFl orthologue increases disease susceptibility.

The virus-induced gene silencing (VIGS) is known to inhibit the expression of genes with high homology and thus, a relatively new approach that has much advantages over mutagenesis in terms of the analysis of genes required for disease resistance when there is functional redundancy of homologous genes (Baulcombe,

1999; Peart et al., 2002).

It has been proved that pepper has 12 homologous chromosomes in contrast to the tetraploid N. tabacum having 24 homologous chromosomes, and the CaPIFl gene is a single copy gene in the pepper genome. According to a blot analysis of N. benthamiana genome DNA, it has been identified that there are two copies of

CaPIFl orthologue.

The results according to the present invention show that the PIF1 -like gene (NbPIFl) of tobacco is an orthologue of CaPIFl which works similarly for defense response to pathogen infection. In the present invention, it has been shown that the silencing of the CaPIFl orthologue decreases resistance to P.s. pv. tabaci. This suggests that the inhibition on the expression of, NbPIFl positively affects multiplication of P.s. pv. tabaci which is a toxic pathogen. Though the gene is essentially required for defense response, it has not yet been proved that TRV-infected plants become to have susceptibility to attack of pathogens, partially or totally.

The VIGS of the orthologue is associated with the reduction in mRNA level of the PIFl-like gene (NbPIFl). The level of NbPIFl transcripts is reduced particularly in TRVr.NbPIFl plants, which indicates that the reduction of the defense response is caused by VIGS. This result may show the similarity between

CaPIFl over-expressed in N. tabacum and VIGSed NbPIFl in N. benthamiana.

In conclusion, according to the present invention, it has been shown that the activity of typical defense response was always presented in tobacco plants over- expressing the CaPIFl gene. Also, according to the present invention, it is understood that the CaPIFl gene is involved in the signal transduction pathway of the defense response of plants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an electrophoretic picture showing the result of genome Southern blotting analysis using CaPIFl cDNA as a probe for total DNAs of hot peppers treated with EcoRI, Hindlll, Xbal and Drall.

FIG. 2 is a blotting picture showing the expression of CaPIFl according to host and non-host disease resistance in hot pepper.

FIG. 3 is a blotting picture showing the expression of CaPIFl according to treatment of chemicals and wound.

FIG. 4 is a GFP picture showing CaPIFl of the present invention is a nucleoprotein, which has a function of transcription regulator. FIG. 5 represents a gene overview, picture and chart showing transcription activity of yeast by CaPIFl .

FIG. 6 represents main structure overviews of expression vector ρMBPl::CaPIFl and inactivated vector ρTRV::NbPIFl.

FIG. 7 is a blotting picture showing constitutive expression of defense 2004/087923

related genes by overexpression of CaPIFl in tobacco.

FIG. 8 represents pictures showing plants with overexpressed CaPIFl have defense mechanism against Pseudomonas syringae pv. Tabaci.

FIG. 9 is a chart showing the repression of bacterial disease by the overexpression of CaPIFl in tobacco.

FIG. 10 represents pictures showing susceptibility to bacterial pathogen susceptibility is enhanced by silencing of CaPIFl homologue^Λ (NbPIFl) in tobacco plants.

FIG. 11 represents the result of blot showing susceptibility to bacterial pathogen is enhanced by silencing of NbPIFl in tobacco plants.

FIG. 12 represents a chart showing susceptibility to bacterial pathogen is enhanced by silencing of NbPIFl in tobacco plants.

DETAILED DESCTIPTION OF THE INVENTION

The present invention will be described in more detail with the following Examples, which are intended to illustrate the examples of the present invention, and therefore they should not be considered to restrict the scope of the present invention.

Example 1: Isolation/Characterization of the CaPFl gene and analysis of CaPFl protein

CaPFl gene was isolated from hot pepper plants and characterized to examine the regulation mechanism in a defense response of plant-pathogen interaction.

Eight-week-old hot pepper plants (C. annuum L. cv. Bukang) grown in a growth chamber under a regimen of 16 hr light and 8 hr dark at 27±2 ^°C and nonhost pathogen (Xanthomonas axonopodis pv. glycienes; Xag 8ra) were used as model systems in this example. 2004/087923

(1) EST sequence DB(see http://plant.pdrc.re.kr) was made randomly in the pepper tissue showing nonhost hypersensitive response by Xag 8ra inoculation. To study the transcription profile, 90 putative transcription factors were isolated from EST sequence DB. The isolated genes were subjected to dot blot analysis by using reverse transcription hybridization.

By method described in 'Lee et al., 2002', genome DNA was isolated from the pepper leaves. 20 g genome DNA was treated by enough TicoRL HincHll, Xbal and Dral. After isolated by 0.7% electrophoresis in agarose gel, treated genome DNA was denatured and transferred onto a Nylon membrane(Amersham, USA) for blotting. Southern blotting was performed as described previously (Church and Gilbert 1984) using full-length CaPIFl cDNA labeled with ³²P-dCTP as a probe.

Dot blot analysis showed induced or repressed genes at altered time after inoculation of Xag 8ra. By many experiment herein, a gene selected from various putative defense response regulation genes (data on other genes were omitted) was named as CaPIFl (Capsicum annuum Pathogen-Induced Factor 1).

Said CaPIFl gene cDNA sequence was analyzed by conventional method (SEQ ID NO: 1). To examine the structure of the CaPIFl cDNA, the 1.1 kb insert DNA of the cDNA was sequenced. CaPIFl cDNA clone contains a single ORF(open reading frame) encoding a putative amino acid sequence(SEQ ID NO: 3) that consists of 261 amino acid residue which has a calculated molecular mass of 27.98 kDa.

Searches of protein databases revealed that the CaPIFl protein contains two C2H2 zinc-fmger DNA-binding domains (CX2CX3FX5LX2HX3H, amino acid

104-124 and 163-183) and the zinc finger motif has QALGGH motif. The

QALGGH motif has been known to participate in plant specific biological process

(Takatsuji, 1998).

Moreover, CaFIFl protein has a basic region which seems to function as 2004/087923

nuclear localization signal (NLS) in N-(³⁵KGKRSKRSR⁴³) and C-(¹⁹⁴PAKKSRL²²⁰) terminal region. The Leu rich region of N terminal region (EXEX₂AXCLX₂, L-box) has been reported that plays a role in protein-protein interaction (Takatsuji, 1998).

In the C terminal region of CaFIFl protein, which is a short hydrophobic region reported (Ohta et al., 2001) to be conserved and to have a function of repressor in plant zinc finger protein, which is L/FDLNL/F(X)PEAF motif; AP2/ERF domain, class II), exists. Furthermore, CaPIFl functions as an active domain for ZPT2-1 and Pszfl (Takatsuji et al., 1992; Michael et al., 1996) or has a Ser-rich region which seems to be a phosphorylation site for post-transcriptional modification.

As a result of sequence homology analysis, C2H2-type zinc finger protein was found to be a transcription factor family (Takatsuji et al., 1992 & 1994) having one or several copies of TFIIIA(CX₂CX₃FX₅LX₂HX₃H) zinc fingers DNA-binding motif which are separated b)^ spacer. Analysis of the predicted amino acid sequence of CaPIFl revealed that the

DNA binding domain of C2H2 zinc finger gene in petunia was highly conserved (Takatsuji et al., 1994). The predicted amino acid sequence of CaPIFl was homologous enough to be 77% identical and 82% similar to that of PetZFP4.

As shown the result of this example, CaPIFl gene isolated from the present invention is suggested to encode the C2H2 zinc finger putative domain and to function as a putative transcription factor.

(2) The tissue-specific expression of the CaPIFl gene was then examined by RNA gel blot analysis. CaPIFl transcripts were abundant in the roots, flowers, fruit, and stem and leaf tissues. For RNA gel blot analysis, total RNA was isolated from the roots, flowers, fruit, and stem and leaf tissues of inoculated hot pepper plant according to the method described in Choi et al., 1996. 20 μg of total RNA from each sample was fractionated by agarose gel electrophoresis containing formaldehyde and transferred onto a Nylon membrane (Amersham, USA). Isolated 2004/087923

RNAs were blotted and the membranes were hybridized with ³²P-labeled full-length CaPIFl cDNA.

CaPIFl transcripts were abundant in the root, flower, and fruit tissues, but were detected a few in stem tissues and almost undetected in leaf tissues. This result shows that the gene expression of CaPIFl is tissue-specific.

(3) The copy number of the CaPIFl gene in the hot pepper genome was examined by DNA gel blot analysis.

The gel blot analysis was performed by the method described in (1). The hot pepper genome DNA was treated with various restriction endonucleases, separated, transferred and hybridized with P-labeled full-length CaPIFl cDNA. As shown in FIG. 1, CaPIFl gene is present as a single copy and shows as strong single or double band patterns on genome DNA blot treated with when four different restriction enzymes (E:EcoRI, Η.:HindIII, X: Xbal and D: Dral). A dual band pattern in the CaPIFl cDNA fragment is an endogenous restriction enzyme site.

Example 2: Analysis of CaPIFl Expression under the Host-incompatible and Nonhost- incompatible condition

(1) Because pathogen Xanthomonas axonopodis pv.vesicatoria race 3(avrBs2, Xav variant 3) which causes bacterial spot pattern disease in pepper is appropriate to compare the resistant ECW-20R (Bs2/Bs2) cultivar and the near- isogenic susceptible ECW (bs2/bs2) cultivar, CaPIFl gene expression was analyzed in ECW near-isogenic pepper cultivar inoculated with the above pathogen (FIG.2). The above pepper cultivars have genetic properties similar to Early Calwonder.

Hot pepper leaves were infiltrated with the two bacterial pathogens by forcefully ejecting the bacterial suspension (1 x 10⁸ cfu ml^"1 in lOmM MgCl₂) with a needless syringe. Control leaves were infiltrated with lOmM MgCl₂. The infiltration was performed by the method described in 'Lee et al., 2002'. 2004/087923

The susceptible plant (bs2/bs2) infiltrated with the bacteria showed no visible response after 36 hours post-infiltration, but the resistant plant (Bs2/Bs2) showed HR cell death on infiltrated leaf tissue within 20 hours post-infiltration.

Total RNA from leaf tissue was isolated at the indicated time points after infiltration (refer to the described method). As shown in FIG. 2, CaPIFl transcripts were strongly induced within 1.5 hours post-infiltration of incompatible Xav variant 3 and as many CaPIFl transcripts as detectable were increased continuously with HR development until 48 hours post-infiltration. PR-1 gene was strongly expressed within 6 hr post-inoculation of Xav variant 3 (FIG.2).

(2) As the method of (1), CaPIFl gene expression was examined in the pepper leaf inoculated with Pseudomonas syringae pv. syringae 61(Pss61; Wheat pathogen) as a non-host bacterial pathogen.

The induction of CaPIFl gene by non-host bacterial pathogen Pss61 was found by hypersensitive resistant response 15~18 hr after inoculation.

As shown in FIG. 2, CaPIFl gene transcription increased rapidly after inoculation, before the onset of the hypersensitive response was visible, and remained elevated until 24 hr after infiltration. In contrast, mock- treated (10 mM MgCl₂) leaf tissues showed increased CaPIFl transcription within 30 min after the inoculation was not induced until 24hr after the inoculation. At the same condition, the PR-1 transcript was first detected within 3~6 hr after inoculation and the PR-1 transcript ion was significantly increased until 24 hr post-infiltration.

The above results show that the CaPIFl transcript was rapidly induced even when the hot pepper was infected with host-incompatible pathogen and the expression of the CaPIFl gene was induced broadly by an elicitor derived from pathogen including the pathogen-related gene expression.

The induction of CaPIFl gene by non-host bacterial pathogen Pss61 in hot pepper plants suggests that the CaPIFl gene has an important role in plant defense responses.

Example 3: Analysis of CaPIFl Expression by Abiotic stress

Induction of the CaPIFl expression in plants was checked under abiotic stress.

(1) In the chemical treatments, plants were sprayed with an aqueous solution with 10% acetone containing 5 mM SA, 100 μM Me-MJ and 5 mM ethephone(Ehte) and an aqueous solution with 10% acetone because it is general that endogenous SA, ET or methyl-jasmonase(MeJA) is accumulated in pathogen- infected plant. And then, the CaPIFl gene expression was examined.

As shown in FIG. 3, the expression of the CaPIFl gene was rapidly induced after the treatment of SA, Ethe and MeJA. PR la, ACC oxidase and PINII gene expressions were followed as a control to abiotic stress. CaPIFl transcripts was detected within 30 min or 1.5 hr after the treatment of SA, Ethe and MeJA and the expression remained elevated until 24 hr post-infiltration. PRl a gene was induced for 12 hr after the treatment of SA and strongly expressed 24 hr after the treatment. ACC oxidase and PINII gene were strongly expressed within 30 min or 1.5 hr after the treatment of Ethe and MeJA.

(2) Northern blot analyses were performed to observe an activation of

CaPIFl gene expression as a response to a wound. For a wound treatment, apical lamina of rosette type of leaf was crushed by spatula to be wounded in about 50% of leaf area.

The accumulation of CaPIFl and PINII transcripts were indicated at 30 min after wound treatment (FIG. 3). Moreover, PINII transcript was activated until 48 hr but CaPIFl gene was decreased within 6 hr after wound treatment.

From these results, CaPIFl gene was suggested to be controlled by various signal pathways mediated by SA and ET/JA, which can play a crucial role in plant defense response. Example 4: CaPIFl as a nuclear protein

By the experiment herein, it was investigated where the CaPIFl protein function. Application of the PSORT program (http://psort.nibb.ac.jp) to the deduced amino acid sequence of CaPIFl also revealed two nuclear localization signal (NLS) sequences in the protein, one in the N-terminal region (³⁵KGKRSKRSR⁴³) and another in the C-terminal region (¹⁹⁴PAKKSRL²²⁰).

Short series alkali amino acid sequence is NLS similar to simian virus 40(SV 40). To investigate the cellular compartmentation of CaPIFl, an in vivo targeting experiment was performed employing CaPIFl fused to the soluble modified form of green fluorescent protein (smGFP) that serves as a fluorescent marker (David and Viestra, 1996). The construct was made as follows.

By the method described in Park at el. 2001, the cellular localization of p35S: : CaPIFl -smGFP construct was examined.

The full-length CaPIFl ORF without the termination codon was prepared by PCR amplification using the CaPIFl cDNA as a template in pBluescript SK- phagemid(Stratagene, La Jolla, CA, USA). Primers used are following.

N terminal primer 5*-GGATCCATGGCACTTGAAGCTTTG-3*(SEQ ID NO: 5) C terminal primer 5'-GGATCG4ATGTTGAAATAATTC-3' (SEQ ID NO: 6)

The PCR amplification products containing BamHI sites were digested with BamHI and then fused into the GFP expression vector p35S-smGFP (David and Viestra, 1996). The empty p35S-smGFP vector was used as a control.

Hot pepper protoplasts were introduced by the fused expression vector and the transient expression of the GFP constructs was performed by the polyethylene glycol-mediated transformation method (Abel and Thellogis, 1994). Green fluorescence images under visible- and UV-light excitation were then captured. GFP fluorescence in the protoplasts introduced by the construct encoding the CaPIFl :smGFP construct protein was exclusively detected in the nucleus (Fig. 4). In contrast, the protoplasts introduced by only the smGFP control showed fluorescence in the entire cytoplasm as well as in the nucleus. Thus, these observations also suggest CaPIFl may function as a transcription regulator in the nucleus. CaPIFl -smGFP fusion protein orienting to the nucleus suggests that CaPIFl is sufficient for putative nuclear targeting sequence after transcription.

Example 5: Analysis of transcriptional activity of yeast by CaPIFl The function of CaPIFl was observed in yeast.

In plants, several transcription factors are known as active repressors (Fujimoto et al., 2000; Ohta et al., 2001). Furthermore, represser domain of transcription factor has ^L/pDLN^L/p(X)P motif(EAF motif), which ERF(class II) and plant zinc finger protein are conserved, and the above motif demonstrates that it can function as a repressor (Ohta, et al., 2001). As a result of comparing amino acid sequences of CaPIFl and class II ERF or zinc finger protein, ^L/_FDLN^L/_F(X)P motif sequence was conserved in C-terminal of each protein.

To observe the transcativation activity of CaPIFl as a repressor or an activator, enzyme transcription activity on fusion DNAs constructs without CaPIFl was analyzed (Clontech, Palo lto, CA, USA). Yeast EGY48 (Clontech, Palo Alto,

CA, USA) was used as a host and manipulated by the standard method described in

"Lee et al., 2002".

LexA DNA-binding domain and construct in-frame were manufactured by inserting BarnHI-Sall insert of CaPIFl derivative into BamHI-Sall site of pLexA (Clontech, Palo Alto, CA).

Not only pLexA and pLexA-GAL4 plasmid as controls but also manufactured plasmids, ρLexA-CaPIFl-F(l-261 aa), ρLexA-CaPIFl-Δ l(l-230aa), pLexA-CaPIFl-Δ2(l-138aa), _PLexA-CaPIFl-Δ3(124-213aa), pLexA-CaPIFl- Δ4(124-261aa) and pLexA-CaPLFl-Δ5(124-230aa), were co-transfoιτned into yeast EGY48(MAT, his3, trpl, ura3, lexA operator-LEU2) with p8op-lacZ encoding lacZ reporter gene under regulation of LexA operator. Clones were cultured in SD(synthesized dextrose) solid medium without Leu at 30^°C for 3 days. Galactosidase activity of yeast clones including LexA constructs was analyzed using ONPG (-nitrophenyl-D-galactopyran) as a substrate by the method described in "Lee et al., 2002". The transcription activity of the above fusion DNA was analyzed by the method of indicating activity of dual reporter genes of LEU2 and LacZ under regulation of LexA operator. Separated region of CaPIFl was expressed as a fusion protein with LexA

DNA-binding domain.

Considering that yeasts are well grown in Leu-deficient solid medium containing X-gal, both fully fused LexA-CaPIFl fusion protein and N-terminal fused LexA-CaPIFl fusion protein strongly activated the expression of LEU2 reporter gene having 8 LexA binding region in promoter.

The transcription activity of LexA-GAL4 protein was used as positive and negative controls.

Single LexA DNA-binding domain or LexA DNA-binding domain fused with C-terminal C2H2 zinc finger domain of separated CaPIFl was not enough for LEU2 reporter gene activation or X-gal activation.

As collectively described in FIG. 5, all 5 kinds of yeast clones were equally grown in solid medium containing Leu (+Leu).

Example 6: Effect of CaPIFl overexpression-enhancing of resistance to pathogen attack

To clarify the role of CaPIFl as a transcription factor in plants, effect of putative C2H2 zinc finger gene overexpression was examined for the study of plant- pathogen interaction in tobacco. (1) Plasmid (pMBPl::CaPIFl) was prepared by introducing Xbal-Kpnl CaPIFl full length cDNA fragment into the plant expression vector pMBPl (modified from pBI121 (Clontech Co. USA)) having 35s promoter in sense orientation under the control of the CaMV 35S promoter(FIG. 6). The constructs were introduced into tobacco cv. Xanthinc using Agrobacterium-mediated transformation. All of the infection experiments were performed on kanamycin- resistant TI transgenic tobacco plants. From the above process, transgenic tobacco plants expressing CaPIFl in sense orientation were generated. 7 sense orientation- expressing TI transgenic plants were selected on the basis of northern blot analysis of Neomycin phosphotransferase (nptll) gene specific oligoprimer, PCR analysis and kanamycin resistance. The above transgenic tobaccos constitutively express CaPIFl under the control of the CaMV 35S promoter.

(2) To observe if the CaPIFl overexpression enhance the resistance to pathogen, the healthy and fully expanded leaves of pMBPl and pMBPl -CaPIFl expressing transgenic lines were challenged with Pseudomonas syringae pv. tabaci which is toxic bacterial fire blight pathogen.

Cells of P.s. pv. tabaci strains were grown in LB medium containing lOOμg/ml ripamficin, and washed by the method of "Thilmony et al., 1995" and then, re-suspended in lOmM MgCl₂ to be OD₆₀o=0.001. The above 2mL of re- suspended bacterial suspension was infiltrated into the healthy and fully expanded leaves using needless syringe.

The bacterial population of the leaves was measured by grinding three leaf discs obtained from the leaves infected by the above strains in lOmM MgCl₂, plating serial dilutions on LB agar medium plates containing lOOμg/ml ripamficin on a daily basis until 6 days after the challenge inoculation. The average bacterial cells was decided by measuring the number of colony forming units per cm , on three leaves per plant, for three plants of each line.

Transgenic tobacco plants expressing CaPIFl were inoculated with Pst, the fire blight pathogen of tobacco. In three independent experiments, the leaves of the CaPIFl transgenic (pMBPl::CaPIFl) plants(hereinafter 35S::CaPIFl plants) showed only mild chlorosis lesions but severe chlorosis lesions appeared on the leaves of the pMBPl plants(wild type). Many chlorosis lesions appeared in 35S::CaPIFl plant, but the density was lower than that of the wild type(pMBPl)(FIG.7, FIG.8). The disease symptoms correlated with the bacterial multiplication and growth on these leaves, considering that the CaPIFl transgenic plants showed 5-fold fewer bacteria at day 4 and 20~50 fold fewer number of bacteria at day 6 after inoculation compared to the wild type plants (pMBPl) (FIG. 9).

Moreover as shown in FIG. 8, CaPIFl transgenic tobacco plants (35S:CaPIFl #2,5,6,9,10,17 and 26) showed excellent resistance to Pseudomonas syringae pv. Tabaci infection compared to the vector transgenic tobacco plant (pMBPl). CaPIFl overexpressing plant showed enhanced resistance to bacterial pathogen.

Example 7: Effect of CaPIFl overexpression -Induction of constitutive expression of PR gene (1) Because 35S::CaPIFl plant showed resistance to bacterial pathogen,

PR- gene or defense-related gene expressions of the 35 S:: CaPIFl plant and the control plant transformed with pMBPl were compared.

To study the functional relation of enhanced resistance by CaPIFl overexpression and activity of defense system, the numbers of SAR markers in 35 S : CaPIF 1 plant were measured.

By RNA blot analysis, it was indicated that PRl, PR3(Chitinase) and R5(Osmotin-like protein) normally induced in SAR development(Glazebrook, 1997; Petersen et al., 2000) were constitutively expressed in 35S:: CAPIF1 plant. PR2(-1 ,3-glucanase), SAR8.2, peroxidase and PRoteinase inhibitor II gene were also strongly activated in 35S::CaPIFl transgenic plant (FIG.7).

The above results suggest that CaPIFl strongly regulate many PR gene expression downregulated in SAR development. It was reported that the increased PR gene expression has correlation with induction of disease resistant response (Malamy et al., 1990; Gaffney et al., 1993; Oldroyd and Staskawicz, 1998; Tang et al., 1999), and suggest that the resistances against diseases were increased by CaPIFl expression.

(2) To investigate CaPIFl expression mediated in details, the full gene expression were compared in 35S::CaPIFl plant and pMBPl plant by the cDNA hybridization method to 4,685 cDNAs microarray fully expressed in the hot pepper plant.

Firstly, the hot pepper microarray was obtained from 4,685 EST clones. PCR products are obtained from plasmid DNA using primer pairs as in the following.

CD Forward primer T7 for three types of hot pepper cDNA library, KS01, KS07 and KS08 library(universal primer)

SEQ ID NO: 7 5'-GTAATACGACTCACTATAGGG-3'

(2) Reverse primer T3 for KS01 and KS08 library(universal primer) SEQ ID NO: 8 5'-AATTAACCCTCACTAAAGGG-3

®' Reverse primer A 1 for KS07 library (universal primer) SEQ ID NO: 9 5'-CGCGTTTGGAATCACTACAGG-3*

The methods such as cDNA microarray preparation, RNA preparation, fluorescent labeling of probe, hybridization, washing and scanning are used with conventional methods (Lashkari et. al., 1997). Spot strength was measured using Axon GenePix PRo 3 image analyzing software, and standardized using the average default elements of corresponding GenePix. After standardization, several quality control methods are applied. Following spots are removed: φ Spots appeared weakly or not by image analyzing software © Spots less than 40μm in size (D Spots that the value subtracted average strength of backgrounds is less than 300 and @ Spots less than 3, median of signal: backgrounds ratio in at least one channel. After quality control, spots whose expressions are more than 3 folds in two-replica array were selected and analyzed.

32%>(1475 over 4685) of the microarrayed hot pepper cDNA was significantly hybridized with 35S:: CaPIFl and pMBPl tobacco seedling cDNA. Only 28 cDNA(1.9%) showed hybridization difference more than 5 folds in 35S::CaPIFl and pMBPl plant. 28 cDNAs were all expressed in 35S::CaPIFl, which suggest that CaPIFl relates to the activity of the above genes(subset).

By database analysis, it was indicated that 13 genes out of the above gene set encoded well-known PR- or wound-induced protein. SAR response (Glazebrook et al., 1997), extensin related to cell wall modification, PR-1 encoding peroxidase related to cell wall cross-linking and 7^JR-2(-l,3-glucanase) were also included in the genes.

Moreover, cystein protease, lipase, HMG-CoA reductase 2, ACC oxidase and ribonucelase genes are considered to participate in plant defense.

Example 8: Effect of NbPIFl silencing-enhancing disease susceptibility

VIGS has been known to repress expression of genes with high homology. Therefore, VIGS results need to be analyzed carefully. In hot pepper, CaPIFl is a single copy gene. The blot analysis of genome DNA of N. benthamiana reveals that PIF1 exists in 2 copies. The above fact corresponds with the character of aneuploid of TV. bethamiana(ln= 19 chromosomes) compared to diploid Nicotiana (ln= 12 chromosomes)(Smith, 1979). Therefore, it is suggested that the PIFl-like gene of tobacco is required for the function of CaPIFl according to the present invention. (1) Therefore, VIGS (Baulcombe, 1999) to a homologue of CaPIFl was performed to investigate the role of Capsicum annum PIF1 in disease resistant response. NbPIFl cDNA(SEQ ID NO: 2) which is, a homologue of CaPIFl, in N benthamiana was required to be identified, because PTGS(Post-transcriptional gene silencing) is dependent on high degree of the sequence homology of the above virus and target DNA (Mueller et al., 1995).

N. benthamiana NbPIFl cDNA was prepared by the method described in "Ratcliff et al., 2001". The pBluscript-SK plasmid containing NbPIFl gene cDNA fragment was treated with BamHI-Kpnl restriction enzyme to obtain the NbPIFl gene cDNA fragment with l.Okb size and to obtain TRV:: NbPIFl by cloning to same site of pTV00(TRV::00) vector (John Innes Center, UK) (FIG.6). The structure of NbPIFl cDNA was examined by analyzing the sequence of 1.1 -kb size insert DNA. The NbPIFl cDNA clone contained a single ORF encoding a deduced protein of 253 amino acid residues and a calculated molecular mass of 27.02 kDa. It was revealed that NbPIFl has two C2H2-type zinc finger domains in the mid-region by database searching. The NbPIFl fragment sequence of N benthamiana was 86% identical to the sequence of the hot pepper CaPIFl and the deduced amino acid sequence of NbPIFl was highly homologous with the CaPIFl sequence, this protein was 80% identical and 84%> similar to CaPIFl.

(2)-l The experiment to investigate the VIGS treatment of NbPIFl gene enhance disease susceptibility to pathogen was performed. TRV derivative was introduced to plants by the agro-infiltration method described in "Ratcliff et al., 2001". The virus was introduced into N benthamiana by the agrobacterium-mediated transient expression of the infection construct from T-DNA of binary plasmid.

Firstly, Agrobacterium was sufficiently proliferated in LB medium containing antibiotics. The culture broth was centrifuged, re-suspended in 10 mM MgCl₂, 10 mM MES and 150 μM acetosyringone, and kept at room temperature for 2 hr. For TRV introduction, separate cultures containing Agrobacterium strain GV3101 (respectively TRV::OO and T_W: NbPIFl constructs) and Agrobacterium strain C58C1 (pBINTRA6) were mixed in a 1:1 ratio (OD₆₀₀=0.5). The above agrobacterium was provided from Dr. David C. Baulcombe of John Innes Center, UK but it is no matter to use Agrobacterium that can be easily obtained.

Independently, N. benthamiana plants were cultured under 16 hr light and 8 hr dark at 24°C. Then the bacterial suspension was infiltrated into the two expanded leaves of 4~5-week-old N. benthamiana plants with a needless syringe.

RNA gel blot analysis was performed to check TRV introduction into plants. According to instruction of manual, total RNA was extracted from the infected plants 2, 3, and 4 days after infection treatment using Trisol-reagent(Gibco-BRL), and then about fractionated 20 g in 1.2% W/V agaros-formaldehyde gel. The fractionated RNA was transferred onto Nytran membrane (Hybond-NX, Amersham phamasia) and then temporary bridges were formed by UV excitation. The membrane was prehybridized, hybridized and then washed according to the known method. PCR amplified NbPIFl fragments were labeled with ³²P to use probe for determining TRV: .NbPIFl. NbPIFl fragments labeled with P were used to analyze endogenous NbPIFl mRNA level on gel blot.

(2)-2 It was examined directly to investigate the effect of VIGS treatment of NbPIFl gene on disease susceptibility to pathogen.

Pseudomonas syringae pv. tabaci (OD₆₀o⁼0.001) which is toxic bacterial pathogen, was inoculated into TRV::00 and TRV: .NbPIFl -induced plants, 12 days after TRV:: 00 and TRV: .NbPIFl solution which is the above bacterial suspension for TRV introducing, were introduced into plants.

As shown in FIG. 10, NbPIFl was required to limit colony formation of P. s. pv. Tabaci which is toxic pathogen, as a result of VIGS treatment on N benthamiana. The bacterial numbers on TRV:: bPIFl '-infected plants were 2-3-fold increased 3 days after inoculation compared to the TRV: :00-infected control plants (FIG. 12).

(2)-3 To check phenotypic data, TRVr. OO and TRV:: NbPIFl -induced N. bethamiana plants infected with P. syringae pv. syringae 61(Pss61) were harvested and the total RNA extracted and examined by RNA gel blot analysis.

As a result, NbPIFl transcript level of TRV::NbPIFl -introduced plants was lower than that of TRV: :00-in rodvιced plants (FIG. 11). Seeing the above result, it can be suggested that the NbPIFl is provided to the disease resistant response against pathogen attack.

INDUSTRIAL APPLICABILITY According to the present invention, useful plants, which have resistance to various biotic and abiotic stresses, can be bred by simple and systematic method.

The novel gene for transcription factor of the present invention, which gives a plant stress resistance, and the protein expressed from the gene are derived from hot peppers which have been used for a long time. Therefore, plants and agricultural products that are safer and transfoπned, can be obtained using the present invention. Furthermore, the present invention provides the resistant mechanism of plants against environmental stresses. Therefore, it can be used as a basis for the studies in the related field and development of technologies.

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Claims

WHAT WE CLAIMED IS:

1. A DNA encoding stress-resistance inducible transcription factor having more than 80% of homology with amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4.

2. The DNA according to claim 1, wherein said DNA has the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

3. A DNA capable of hybridizing with the DNA of claim 2 under a strict condition.

4. The DNA according to claim 1, wherein said stress is viral, bacterial, fungal pathogen or abiotic stress.

5. A plant stress resistance inducible transcription factor protein which is encoded by the DNA of any one of claims 1 to 4.

6. The protein according to claim 5, wherein said transcription factor protein is CaPIFl having amino acid sequence of SEQ ID NO: 3 or NbPIFl having amino acid sequence of SEQ ID NO: 4.

7. The protein according to claim 5, which has C2H2-type zinc finger motif.

8. An expression vector having the DNA of any one of claims 1 to 4.

9. A plant cell transformed with the expression vector of claim 8.

10. A stress resistant plant or its seed transformed with the expression vector of claim 8.

11. A method for producing a stress resistant plant which comprises introducing the DNA of any one of claims 1 to 4 into a plant.

12. A method for controlling stress resistance of a plant which comprises changing expression of the stress resistance inducible transcription factor protein of claim 5.